This paper describes a new, simple, and sensitive method for detecting two fluoroquinolones: enrofloxacin and its metabolite ciprofloxacin, which are widely used as drugs for humans and animals. We utilized gold nanoparticles (AuNPs) and laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS) with a matrix-free format. An antibody for the drug was immobilized on a chip based on self-assembled monolayers (SAMs) on gold, and AuNPs were decorated with the drug along with a large excess of small molecules, called amplification tags (Am-tags). In this strategy, target drugs in solution bound to the antibody on the chip compete with the AuNP-immobilized drugs. The presence of targets was verified by the amplified LDI-TOF MS signals of Am-tags on AuNPs.